This research proposal is intended to provide predoctoral and postdoctoral training to develop the necessary skills for a career as an independent investigator in cancer biology. The long-term research focus is development of effective therapy for pancreatic cancer (PC) through 1) determining the contribution of cancer stem cells (CSCs) to PC progression and metastasis, 2) understanding the mechanism of CSC maintenance and CSC- mediated drug resistance, 3) identifying genetic, epigenetic, and metabolic factors essential for CSC maintenance with the aim of identifying novel epigenetic and metabolic targets that can be exploited for combinatorial therapy against PC. The objective of my dissertation research (F99 phase) is to define the role of Pancreatic Differentiation 2 (PD2) in pancreatic CSCs and CSC-mediated PC progression, with the goal of deciphering the mechanism of PD2-dependent CSC maintenance. PD2 is a ubiquitous multifunctional protein, a core component of human RNA Polymerase II-Associated Factor 1 complex (PAF1C) that functions in transcription elongation and mRNA processing. We discovered that PD2 is a novel pancreatic CSC marker and mediates drug resistance of CSCs. Knowledge of the molecular mechanism of PD2-dependent CSC maintenance and drug resistance is critical. We have recently made several discoveries relevant to this concept. First, knockdown of PD2 significantly reduces the levels of established CSC and self-renewal markers. Importantly, PD2 depletion significantly reduces tumor burden in vivo. Moreover, RNA-sequencing and transcription factor PCR array analyses revealed that several stemness and metastasis genes were significantly downregulated following PD2 depletion. Based on aforementioned information and additional data, we hypothesize that PD2 functions as a master-regulator of stem cell maintenance and thereby mediates PC progression. Our research will utilize high throughput genomic techniques such as chromatin immunoprecipitation sequencing and RNA-sequencing in presence and absence of PD2 to define downstream targets of PD2, and identify the pathway for PD2-dependent maintenance of pancreatic CSCs. We will also determine the role of PD2 in CSC-mediated PC progression using a novel CRISPR- based PD2 knockout model crossed with KPC model of PC progression. To expand upon the future research direction and to build the foundation for independence, I will pursue postdoctoral training in cancer metabolism. The goal of the proposed K00 postdoctoral training is to gain expertise on :1) the current metabolomic strategies; 2) understand the mechanisms that promote acquisition of different metabolic programs by CSCs and differentiated tumor cells; 3) contribution of metabolism to CSC-mediated drug resistance; and 4) development of novel combinatorial therapeutics based on metabolic targeting to treat cancer. Ultimately, the proposed F99/K00 training will provide a strong intellectual foundation for R01 funding that will establish my independence and will provide professional training in the skills required to be an effective PI and mentor.